Genetically Engineered Mouse Models of Ovarian Cancer and Their Utility in Drug Discovery

Sanja Šale1

1 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.11
DOI:  10.1002/0471141755.ph1411s45
Online Posting Date:  June, 2009
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Abstract

Ovarian cancer is the fourth most common cancer in women and the most lethal gynecological malignancy. The high mortality rate is attributable to the asymptomatic nature of the early stage of the disease, the lack of reliable screening tests, and the development of drug resistance. Approximately 90% of ovarian cancers are thought to originate from the ovarian surface epithelia (OSE). Development of in vivo experimental models that accurately recapitulate genetic events that occur during human epithelial ovarian cancer (EOC) initiation and progression is crucial for a better understanding of EOC pathogenesis, identification of early disease markers, and development of more effective therapy. Historically, one of the most challenging problems in developing genetically engineered mouse models (GEMMs) of EOC has been the lack of tissue‐specific promoters that regulate transgene expression exclusively in adult OSE cells. Recent improvements in gene delivery technology have greatly accelerated development of GEMMs of EOC. This unit describes two distinct methods of transforming OSE cells in GEMMs and the potential applications of these models in oncology drug discovery and development. Curr. Protoc. Pharmacol. 45:14.11.1‐14.11.14. © 2009 by John Wiley & Sons, Inc.

Keywords: epithelial ovarian cancer (EOC); genetically engineered mouse models (GEMMs); ovarian surface epithelium (OSE); specific genetic lesions; RCAS/TVA gene delivery system; Ad‐CRE

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: RCAS/TVA‐Based Ex Vivo Gene Delivery Model System for Rapid Generation of Genetic Lesions in OSE Cells
  • Basic Protocol 2: Orthotopic Delivery of Cre Recombinase in Cre‐loxP Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: RCAS/TVA‐Based Ex Vivo Gene Delivery Model System for Rapid Generation of Genetic Lesions in OSE Cells

  Materials
  • DF‐1 cell line, an immortalized line of chicken fibroblasts (ATCC, cat. no. CRL‐12203, name UMNSAH/DF‐1)
  • DF‐1 cell line growth medium (see recipe)
  • Plasmids (Addgene) including:
    • RCAS‐K‐ras (activated mouse K‐rasG12D)
    • RCAS‐myrAkt (myristoylated and HA‐tagged mouse Akt1)
    • RCAS‐myc (human c‐myc)
  • Freezing medium (see recipe)
  • Liquid nitrogen
  • OSE cell growth medium (see recipe)
  • 3‐ to 6‐week‐old K5TVA/p53−/− transgenic female mice (contact Silvio Gutkind at gutkind@dir.nidcr.nih.gov)
  • CO 2 gas
  • 70% ethanol
  • 1× PBS
  • Trypsin (e.g., Invitrogen)
  • 6‐ to 8‐week‐old female nude mice (nu/nu; Charles River Laboratories)
  • Autoclave for sterilizing instruments
  • 100‐mm and 30‐mm tissue culture plates
  • Transfection kit (e.g., Lipofectamine 2000, Invitrogen)
  • Filtered pipet tips
  • Cryovials
  • 0.8‐µm syringe filter (low protein binding, Millipore)
  • Transparent chamber for mice
  • CO 2 gas cylinder
  • Absorbent, plastic‐backed pad
  • Surgical equipment including:
    • Disposable scalpels
    • Surgical scissors
    • Tissue forceps
    • Surgical scalpels
  • 1‐ml syringes
  • 25‐G needles
NOTE: Growth medium, trypsin, and PBS should all be prewarmed to 37°C unless indicated otherwise.

Basic Protocol 2: Orthotopic Delivery of Cre Recombinase in Cre‐loxP Mice

  Materials
  • Virucidal disinfectant (e.g., Clorox or Lysol)
  • 6‐ to 8‐week‐old p53loxP/loxP/Rb1loxP/loxP transgenic female mice (contact Alexander Yu Nikitin at an58@cornell.edu) or 6‐ to 8‐week‐old LSL‐K‐RasG12D/+ PTEN loxP/loxP transgenic female mice (contact Daniela Dinulescu at ddinulescu@rics.bwh.harvard.edu) or 8‐ to 10‐week‐old ApcloxP/loxP/PTENloxP/loxP transgenic female mice (contact Kathleen R. Cho at kathcho@umich.edu)
  • 2.5% Avertin (Tribromoethanol)
  • Betadine solution
  • 70% ethanol
  • Warm water bottles
  • Ad‐Cre, replication‐deficient recombinant adenovirus expressing CRE recombinase (Ad5CMVCRE) under the CMV promoter (commercially available, e.g., University of Iowa Gene Transfer Vector Core, http://www.uiowa.edu/∼gene/ or University of Michigan Vector Core, http://www.med.umich.edu/vcore)
  • 1× PBS
  • Post‐operative analgesic (e.g., Buprenorphine; Bedford Labs)
  • 10% bleach solution
  • Class II biosafety cabinet
  • Absorbent, plastic‐backed pad
  • Protective clothing including:
    • Shoe covers
    • Laboratory coat
  • Animal cages
  • Sharps container
  • Red biowaste disposal bags
  • 1‐ml syringe
  • 25‐G needle
  • Animal hair clippers
  • Surgical instruments (VWR or Fine Science Tools) including:
    • Dissecting scissors
    • Tissue forceps
    • Disposable scalpels
  • Stereomicroscope
  • 10‐µl Hamilton syringe equipped with a 30‐G needle
  • Absorbable suture stitch (e.g., Poliglecaprone 25, Ethicon)
  • Sterile stainless wound clip
CAUTION: Adenovirus has limited pathogenic potential and requires BL2 handling during transportation, preparation, and inoculation. The entire procedure should be performed in a class II biosafety cabinet (BSC). Diluted adenovirus should be transported from the laboratory to the animal facility in a shatterproof and sealed primary receptacle, placed in a secondary container containing absorbent material, in accordance with CDC guidelines (BMBL 4th edition, page 219).NOTE: All surgical procedures should be performed in a disinfected work area using aseptic techniques and sterile instruments. Personnel performing the surgery should wear protective garments (cap, facemask, goggles, gloves, gown, and booties).NOTE: 2.5% Avertin is not commercially available but can be prepared by mixing 2,2,2 Tribromoethanol and amylene hydrate (Sigma‐Aldrich). Check institutional IACUC protocols for guidance on how to prepare the solution.
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Figures

Videos

Literature Cited

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